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Brain Res Mol Brain Res. Author manuscript; available in PMC 2018 February 08.
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Published in final edited form as:

Brain Res Mol Brain Res. 2005 August 18; 138(2): 191–197. doi:10.1016/j.molbrainres.2005.04.009.

Valerian extract and valerenic acid are partial agonists of the 5-

HT5a receptor in vitro
Birgit M. Dietza,b, Gail B. Mahadya,b,*, Guido F. Paulib, and Norman R. Farnsworthb
aDepartment of Pharmacy Practice, College of Pharmacy, University of Illinois, 833 S. Wood
Street, Rm 122, MC 886, Chicago, IL 60612, USA
bNIH Center for Botanical Dietary Supplements Research, University of Illinois at Chicago,
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College of Pharmacy, 833 S. Wood Street, Chicago, IL 60612, USA

Abstract
Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription
drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are
also used for the treatment of insomnia and restlessness. To determine novel mechanisms of
action, radioligand binding studies were performed with valerian extracts (100% methanol, 50%
methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and
GABAA receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong
binding affinity to the 5-HT5a receptor, but only weak binding affinity to the 5-HT2b and the
serotonin transporter. Subsequent binding studies focused on the 5-HT5a receptor due to the
distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the
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sleep–wake cycle. The PE extract inhibited [3H]lysergic acid diethylamide (LSD) binding to the
human 5-HT5a receptor (86% at 50 μg/ml) and the DCM extract inhibited LSD binding by 51%.
Generation of an IC50 curve for the PE extract produced a biphasic curve, thus GTP shift
experiments were also performed. In the absence of GTP, the competition curve was biphasic (two
affinity sites) with an IC50 of 15.7 ng/ml for the high-affinity state and 27.7 μg/ml for the low-
affinity state. The addition of GTP (100 AM) resulted in a right-hand shift of the binding curve
with an IC50 of 11.4 μg/ml. Valerenic acid, the active constituent of both extracts, had an IC50 of
17.2 AM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-
HT5a receptor.

Keywords
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Circadian rhythm; Insomnia; Serotonin; 5-HT5A; Valerian; Valerenic acid

1. Introduction
Insomnia is considered the most frequent sleep complaint and affects nearly all populations
throughout the world, particularly the elderly [28]. Menopausal women often suffer from
sleep disturbances with frequent awakenings, likely due to disturbed expression of daily

*
Corresponding author. Department of Pharmacy Practice, Rm 122, College of Pharmacy, University of Illinois, 833 S. Wood Street,
MC 886, Chicago, IL 60612. Fax: +1 312 413 5894.
 Dietz et al. Page 2

biological rhythms [4,30]. Some of the more commonly used hypnotic drugs to treat
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insomnia are benzodiazepines, which are associated with adverse events such as tolerance,
dependence, and morning sleepiness [28]. Many menopausal women prefer not to use
benzodiazepines but use valerian preparations instead, and report that these products are well
tolerated, effective for the treatment of insomnia, while not inducing impairment of
vigilance or cognitive and psycho-motor performance [17,29,30].

Extracts from the roots of valerian (Valeriana officinalis L., Valerianaceae) have long been
used in alternative medicine for the treatment of insomnia and are the most well recognized
herbal sedatives worldwide. Approximately 15 controlled clinical trials have assessed the
efficacy of various valerian extracts, and the German Commission E has approved its use for
the treatment of restlessness and sleeping disorders [4]. Numerous clinical trials support the
use of valerian and have demonstrated an improvement in sleep latency and quality in both
healthy volunteers and patients with sleep disorder [4,29,33]. Currently, there is no scientific
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agreement on the mechanism of action of valerian's sedating activity or the compounds

responsible. Many potential mechanisms for the pharmacological activity of valerian have
been proposed, including agonistic activities on the GABA, adenosine, barbiturate, and
benzodiazepine receptors [34,40,46]. However, the concentrations used in these
investigations were extremely high in the older reports and more recent reports put some of
these data into question [12,40].

Although serotonin is well known to modulate a variety of physiological and behavioral

processes including sleep– wake cycles, and circadian rhythms, the effect of valerian on
serotonin receptor binding has not been completely elucidated [9,24]. Serotonin (5-HT, 5-
hydroxytryptamine) impacts numerous sensory, motor, and cortical functions by activating
multiple 5-HT receptor subtypes [9,24,35]. Abnormalities of these receptor systems have
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been implicated in many psychiatric disorders including anxiety, depression, as well as

disorders of cognition, stress, and sleep [9,28,35]. Serotonin was initially thought to be a
true neuromodulator of sleep because the destruction of 5-HT neurons of the raphe system
or the inhibition of 5-HT synthesis with p-chloro-phenylalanine induced severe insomnia
that could be reversed by restoring 5-HT synthesis [2]. More recent experiments suggest that
the release of 5-HT during the sleep–wakefulness cycle initiates a cascade of genomic events
in some hypnogenic neurons located in the preoptic area and the neighboring
suprachiasmatic nucleus including vasoactive intestinal polypeptide and GABAergic
mechanism [1,3,9,15,28,35].

Our previous work has shown that dichloromethane and petroleum ether extracts of valerian
bind weakly (51% at 50 μg/ml) to the 5-HT2b receptor but have significant binding (80%) at
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the 5-HT5a receptor [12]. When compared with other serotonin receptors, little is known
about the 5-HT5a receptor, but it is believed to be involved in circadian (sleep–wake)
rhythms, anxiety, and explorative behavior [15,18,20,42]. Distribution of the 5-HT5a
receptor is widespread throughout the rat brain, but the receptor concentrations are
particularly intense in the suprachiasmatic nucleus of the hypothalamus, the site of the
biological clock that drives circadian rhythms. Although the involvement of the 5-HT5a
receptors is a more recent hypothesis, support for the 5-HT5a-mediated function in the SCN
has been shown by strong distinct immunoreactivity in three neural components of the

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 Dietz et al. Page 3

circadian timing system—the intergeniculate leaflet, median raphe, and dorsal raphe
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nucleus, in addition to the SCN [42].

Considering that our previous work indicated that valerian extracts bind predominantly to
the 5-HT5a receptor, the purpose of this work was to determine if the valerian extract acts as
an agonist of this receptor. To this end, bioassay-guided fractionation of the DCM and PE
extracts to isolate and identify the chemical constituents responsible for this activity was also
performed.

2. Materials and methods

2.1. Materials and reagents
All chemicals and reagents were purchased from Fisher (Hanover Park, IL) or Sigma (St.
Louis, MO) unless otherwise indicated. All cell culture media were obtained from Life
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Technologies (Carlsbad, CA). FBS was acquired from Atlanta Biologicals (Norcross, GA).
[3H]Lysergic acid diethylamide (LSD), [3H]hydroxytryptamine (5-HT), and [3H]-8-
hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) were obtained from NEN Life Science
Products (Boston, MA). 5-HT5a membranes were purchased from Perkin-Elmer (Shelton,
CT), while valerian reference compounds (valerenic acid, hydroxyvalerenic acid, and
acetoxyvalerenic acid) were obtained from ChromaDex (Santa Ana, CA). The roots and
rhizomes of valerian were obtained and identified from the field station of the University of
Illinois at Chicago. Voucher specimens have been deposited at the University of Illinois at
Chicago, College of Pharmacy.

2.2. Extraction and isolation

Valerian extracts for screening purposes were prepared from dried powdered underground
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parts of V. officinalis L. by successively extracting 100 g dried powdered material each time
three times at 500 ml of the following solvents and an ultraturrax at ambient temperature: (1)
dichloro-methane (DCM), (2) petroleum ether (PE), (3) methanol (MeOH), and in the end
with methanol 50% (MeOH 50%). Moreover, an extract was prepared by extracting 20 g
dried powdered underground parts of V. officinalis only with methanol using the same
procedure. Bioassay-guided fractionation: 1300 g of V. officinalis underground parts was
exhaustively extracted in two parts each time three times at 1500 ml petroleum ether and an
ultraturrax at ambient temperature. The extraction yield was 1.3% and 1.4%, respectively.
The dried petroleum ether extract was separated by silica gel vacuum column
chromatography with a petroleum ether and chloroform gradient (2.5% to 5.0% steps). The
active fraction (13.3 g) was further fractionated by a second silica gel vacuum column with
hexane and ethyl acetate as gradient (2.5% to 5.0% steps). Valerenic acid (4.2 mg) was
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isolated from subfraction 5 by semipreparative HPLC with a water/acetonitrile gradient (75–

85% acetonitrile in 28 min). Besides valerenic acid, the separation resulted in two compound
mixtures (0.9 mg and 1.4 mg) and one isomeric mixture (1.4 mg). The content of valerenic
acid was determined by HPLC using the method with an external standard curve for
valerenic acid [7]. RP-Column a 1:1 combination of the dichloroform (DCM) and PE
extract, a 1:2 mixture of the methanol (MeOH) and MeOH 50% and a 100% MeOH.

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2.3. Serotonin receptor binding assays

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Initial radioligand binding studies were performed by MDS Pharma Services (Bothell, WA)
as previously described for serotonin receptor subtypes 1A, 1B, 2B, 2C, 3, 5A, 6, 7,
serotonin transporter [5,6,8,23,31,32,37–41,43–45,47]. Significant binding affinity was only
observed at the 5-HT5a receptor (>50%) for the PE and DCM extracts at a concentration of
50 μg/ml [12]. Similar results were reported by Schumacher et al. [40]. Therefore, this
investigation focused only on the 5-HT5a receptor assay and GTP shift experiments.

For the 5-HT5a receptor binding experiments, additional assays were performed with minor
modifications using human 5-HT5a receptor expressed in Chinese hamster ovary (CHO-K1)
cell membranes. The human 5-HT5a-transfected CHO-KI cells were grown in Ham's F12
media supplemented with 10% FBS, MEM sodium pyruvate (1 mM), gentamicin (50 mg/
ml), and penicillin/streptomycin (50 U/ml). Cells were scraped from culture dishes at full
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confluence. These cells were homogenized and centrifuged twice at 12,000 ×g for 20 min.
The pellets were dissolved in TEM buffer (75 mM Tris, 1 mM EDTA, 12.5 mM of MgCl2,
pH 7.4) and stored at −80°C. Protein concentrations were determined by the Lowry method
using bovine serum albumin as standard. All preparations were kept on ice. [3H]LSD (1.64
nM) incubated at 37 °C for 60 min in incubation buffer (50 mM TRIS (base), 10 mM
MgCl2·6H2O, 0.5 mM EDTA). After a 1-h incubation at 37°C, the mixtures for both
receptors were filtered over 934-AH Whatman filters that had been presoaked in 0.5%
polyethylenimine (PEI) and washed two times in ice-cold 50 mM Tris buffer (pH 7.4) using
a 96-well Tomtec-Harvester (Orange, CT). Each filter was dried, suspended in Wallac
microbeta plate scintillation fluid (Perkin-Elmer Life Sciences, Boston, MA), and counted
with a Wallac 1450 Microbeta liquid scintillation counter (Perkin-Elmer Life Sciences,
Boston, MA). 5-Hydroxytryptamine (serotonin, 5-HT) (250 nM) was used to define
nonspecific binding, which accounted for <10% of the total binding. The percent inhibition
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of [3H]-LSD binding to the 5-HT5a receptor was determined as [1 – (dpmsample – dpmblank)/

(dpmDMSO – dpmblank)] × 100. The inhibition of [3H]-LSD binding (%) of the sample was
calculated in comparison with the inhibition of 1 μM 5-HT (100%). For the most potent
compounds, IC50 values were determined by evaluation of the percent inhibition of [3H]-
LSD binding in a number of serial dilutions. The data represent the average of triplicate
determinations.

2.4. GTP shift experiments

Inhibition of binding of [3H]LSD (68.2 Ci/mmol, Perkin-Elmer) by the petroleum ether
extract was measured in the presence and in the absence of 100 μM GTP. 5-HT (500 μM)
was used to define nonspecific binding [8,40]. The assay was carried out under the same
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conditions as described for the assay above.

2.5. Data analysis

The data obtained in our laboratory represent the average ± SD of at least triplicate
determinations. Curve fitting was performed using Akaike's Information Criteria (AICs),
GraphPad Prism version 4.01 for Windows, GraphPad Software, San Diego CA,
www.graphpad.com. Kd, IC50, and Ki values were determined using the same program.

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 Dietz et al. Page 5

3. Results
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Extracts of valerian (100 MeOH, 50% MeOH, DCM and PE) were initially screened to
determine if these extracts contained any potential ligand(s) of the melatonin, serotonin,
benzodiazepine, glutamate, and GABAA receptors, or serotonin transporter [12]. No
significant binding was found for any of the extracts at the 5-HT1A, 1B, 2C, 3, 6, 7,
melatonin, benzodiazepine, or glutamine receptors. Only the PE and the DCM extracts (50
μg/ml) were active in the 5-HT2b, 5-HT5a receptor assays, and the serotonin transporter
assay. A combination of the PE/DCM extracts bound to the serotonin 5-HT2b (51% binding),
serotonin transporter (53% binding), and in the 5-HT5a receptor binding assay (80%
binding) at a concentration of 50 μg/ml (Fig. 1). When tested separately, the PE extract
exhibited 86% binding at the 5-HT5a receptor as compared with a 51% binding of the DCM
extract at a concentration of 50 μg/ml. None of the other extracts tested were active (data not
shown). The 100% methanol and 50% methanol extracts were not active in any of the
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receptors or transporter tested.

Due to the dense distribution of the 5-HT5a receptor to areas in the brain responsible for
circadian timing and the sleep–wake cycle, the binding of the PE extract to the 5-HT5a
receptor was investigated in further detail. To validate this method, serotonin (5-HT) was
tested as an inhibitor [3H]LSD binding to the 5-HT5a receptor. A binding assay was
established and validated by creating a saturation curve with [3H]LSD. Experimentally
generated Ki values for [3H]LSD were consistent with the literature (Ki = 1.8 nM). The PE
extract displaced the [3H]LSD from the 5-HT5a receptor with an IC50 curve that depicted a
biphasic shape and best fit a two-site competition equation using the Akaike's Information
Criteria (GraphPad Prism 4.01 for Windows). The PE extract displaced radioligands from
the 5-HT5a receptor with an IC50 of 15.7 ng/ml (Ki: 9.48 ng/ml) for the high-affinity state
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and 27.7 μg/ml (Ki: 16.7 μg/ml) for the low-affinity state (Fig. 2). Since the 5-HT5a receptor
belongs to the G-protein-coupled transmembrane proteins, GTP converts two classes of
binding sites to a single lower affinity class of sites and leads to a right shift in case of an
agonistic activity at the receptor. The IC50 curves for the PE extract in the absence and
presence of GTP are depicted in Fig. 3. GTP can cause an uncoupling of the receptor from
the G-protein leading to a shift of the receptor from the high- to the low-affinity state for
agonists [40]. The addition of GTP resulted in a rightward shift of the PE extract binding
curve to a low-affinity state or monophasic with an IC50 of 11.4 μg/ml (Ki = 6.88 μg/ml).
These data suggested that chemical components of the PE valerian extract function as an
agonist of the human 5-HT5a receptor.

In order to identify the active chemical compounds from valerian that bound to the 5-HT5a
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receptor, both the PE and DCM extracts were fractionated and pure compounds were
isolated. In contrast to the methanol extracts tested, both the DCM and PE extracts contained
valerenic acid (Fig. 3), with the PE extract characterized by its high valerenic acid content.
In addition to valerenic acid, the PE extract also contained trace amounts of acetoxy-
valerenic acid (Fig. 3) and a mixture of valepotriates. Hydroxyvalerenic acid (Fig. 3) was not
found in the PE but did occur in the DCM extract. To isolate the active 5-HT5a receptor
binding ligand from the PE extract, bioassay-guided fractionation was conducted leading to
several active fractions, all containing valerenic acid. Receptor binding comparisons of the

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three major sesquiterpenes, valerenic acid, hydroxyl valerenic acid, and acetoxy-valerenic
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acid, confirmed that valerenic acid was the active constituent (see Fig. 4 and Table 1). The
semipreparative HPLC fractionation of the active fraction yielded valerenic acid as the major
active compound, as well as valepotriate isomeric mixture, which also exhibited some
activity (62%) at a concentration of 50 μg/ml in the 5-HT5a receptor binding assay. Valerenic
acid exhibited significant affinity for the 5-HT5a receptor (80% at 50 μg/ml), with an IC50
value for valerenic acid of 17.2 μM (Ki: 10.7 μM).

To correlate the valerenic acid content of various valerian extracts with the 5-HT5a receptor
binding activity, we tested the PE and DCM extracts of three different batches of plant
materials. The results clearly demonstrate that there is a direct correlation between the
valerenic acid content of these extracts and the 5-HT5a receptor binding activity (Table 1).
Extracts prepared from valerian roots having a high concentration of valerenic acid were
associated with a higher significant affinity for the 5-HT5a receptor.
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4. Discussion
Valerian has been used as a sedative for the treatment of insomnia and restlessness since the
second century [26, 27,29,33]. Currently, valerian is approved in Europe as a sedative by the
German Commission E. Approximately 15 controlled clinical trials have been published to
date and suggest that treatment with valerian extracts improves sleep structure and sleep
perception of insomnia in healthy patients and in patients suffering from sleep disorders,
without producing the traditional sedative side effects [10,13–16,21,22,25,45]. Valerian
extracts have also been used for the relief of sleep disorders associated with aging and
menopause [19,43]. Interestingly, Vorbach et al. [45] reported that approximately 2–4 weeks
of therapy with valerian is needed to achieve significant improvements in sleep disturbances.
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Clinically, valerian does not cause dependency, and there does not appear to be any additive
effects with alcohol [4]. Thus, the mechanism by which valerian exerts its sedative activity
may be unlike other sleep aids.

Many potential mechanisms for the pharmacological activities of valerian have been
proposed, including agonistic activities on the GABA, adenosine, barbiturate, and
benzodiazepine receptors [4,29,48]. However, newer studies have questioned these data
[12,40]. Schumacher et al. [40] found no significant binding of valerian compounds to either
benzodiazepine or GABAA receptors in concentrations up to 100 μM. These data are
supported by our previous work [12]. Furthermore, the concentrations of valerian extracts
used in at least one study were extremely high [48]. This study reported that valerian
extracts bound to the GABA receptor in vitro; however, the concentration of valerian extract
used was 3 mg/ml (29.6%) [48]. Thus, considering the concentration used and the low
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binding affinity, the results are questionable.

In contrast to other plausible mechanisms, the effects of valerian on the serotonin receptor
have not been fully investigated. Serotonin is well known to modulate sleep– wake cycles
and circadian rhythms in the human brain via a G-protein-coupled receptor family [24,28].
Reports on the binding affinity of valerian to serotonin receptors are few. In one previous
investigation, several compounds isolated from a valerian extract were reported to have no

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significant affinity for the 5-HT1a receptor [40]. This is also in agreement with our work, as
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none of our valerian extracts bound to the 5-HT1a receptor in concentrations up to 50 μg/ml
[12]. Only the DCM and PE extracts of valerian had high binding affinity for the 5-HT5a
receptor. Binding of valerian extracts to the 5-HT5a receptor has not been previously
reported, and thus may represent a new mechanism of action. Valerenic acid, one of the
major constituents of valerian, is the primary active compound; however, a synergistic
mechanism involving other compounds present in the PE extract cannot currently be ruled
out. Valerenic acid was present in both the DCM and PE extracts of valerian; however, the
PE extracts contained a higher concentration of valerenic acid than the DCM extracts, thus
explaining the higher binding affinity to the 5-HT5a receptor. Since the binding affinity of
the extracts was dependent on the concentration of valerenic acid, and neither methanol nor
50% methanol extracts contained valerenic acid, they were not active in this assay.

Due to of the association of G-proteins with the 5-HT5a receptor, GTP binding studies were
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used to determine if valerian extracts exhibited agonist or antagonist activity. While agonists
stimulate the binding of GTP to the G-protein, neutral antagonists have no effect, and
antagonists with inverse agonistic activity reduce GTP binding. Thus, addition of GTP
induces an uncoupling of the receptor from the G-protein leading to a shift of the receptor
from the high- to the low-affinity state for agonists [40]. In our experiments, the binding
curve for the PE extract was biphasic in the absence of GTP, and addition of GTP resulted in
a rightward shift of the PE extract binding curve to a low-affinity state. These data support
the hypothesis that valerian extract and valerenic acid are partial agonists of the 5-HT5a
receptor, and suggest an entirely novel mechanism to explain the sedative effects of valerian.
The 5-HT5a receptor is expressed in many brain regions, including several important neural
components of the circadian timekeeping system, namely the suprachiasmatic nucleus
(SCN, in the hypothalamus), the intergeniculate leaflet, the dorsal raphe nucleus, and the
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median raphe nucleus [11,15,36]. The SCN is thought to tag the 24-h endogenous circadian
clock that regulates sleep and wakefulness [11]. Thus, it has been proposed that the 5-HT5a
receptor may play a role in the serotonergic regulation of circadian timekeeping
[15,18,36,42]. Confirmation of this mechanism of action should be performed in vivo using
5-HT5a receptor knockout mice [20].

Acknowledgments
This study was partly funded by NIH Grant P50 AT00155 jointly provided to the UIC/NIH Center for Botanical
Dietary Supplements Research by the Office of Dietary Supplements, National Institute for General Medical
Sciences, Office for Research on Women's Health, National Center for Complementary, and Alternative Medicine.
The contents are solely the responsibility of the authors and do not necessarily represent the views of the funding
agencies.
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Fig. 1.
Percent binding affinity of the dichloromethane/petroleum ether extract combination, the
dichloromethane extract, and petroleum ether extract to the 5-HT5a receptor. Extracts were
tested at concentrations of 50 μg/ml in duplicate.
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Fig. 2.
Binding of the PE extract of valerian to human 5-HT5a-transfected CHO membranes in the
presence of [3H]LSD and in the presence or absence of GTP (100 μM). GTP results in a
right-hand shift of the IC50 curve, indicating that the extract acts as an agonist at the 5-HT5a
receptor.
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Fig. 3.
Major sesquiterpenes present in the PE and DCM valerian extracts. (1) Valerenic acid, (2)
acetoxyvalerenic acid, and (3) hydroxyvalerenic acid.
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Fig. 4.
Binding affinity of the sesquiterpenes valerenic acid, hydroxyvalerenic acid, and
acetoxyvalerenic acid to the 5-HT5a receptor tested at a concentration of 50 μg/ml, n = 3.
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Table 1

Correlation between the valerenic acid concentration and the 5-HT5a receptor binding activity of the PE- and
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DCM extracts of different V. officinalis plant samples

Extract Milligram valerenic acid/100 mg extract Activity in 5-HT5a assay, percent specific binding

Petroleum ether (PE) extracts were tested at a concentration of 20 μg/ml

PE 1 7.33 64.93
PE 2 8.21 76.02
PE 3 6.23 40.4
Dichloromethane (DCM) extracts were tested at a concentration of 50 μg/ml
DCM 1 2.88 70.88
DCM 2 3.39 71.38
DCM 3 1.94 46.83

The valerenic acid content was determined by external calibration (r = 0.96), every data point was at least obtained by triplicate determination.
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